In this laboratory, the following proteolytic enzyme activities have been identified and fractionated from the soluble proteins of rat and calf lenses: leucine aminopeptidase, arylamidase and neutral proteinase. We have determined the in vitro relative rates of hydrolysis of calf lens crystallins by calf lens neutral proteinase to be beta L greater than alpha greater than beta H greater than gamma-crystallin. The purified B chains of alpha-crystallin were found to be hydrolyzed at a greater rate than the A chains or unfractionated alpha-crystallin. Using alpha-crystallin as substrate, we have shown that the enzyme cleaves at the carboxyl side of the aliphatic amino acids valine, isoleucine, leucine and alanine. We propose to further purify and characterize the lens neutral proteinase. The lens neutral proteinase preparation currently used is associated with apha-crystallin polypeptides. Chaotropic agents, proteolytic digestion and extremes of pH and temperature will be used to disrupt this association. Lens protein products of hydrolysis will be identified and the relationship of in vitro products of hydrolysis to in vivo degradation products will be determined. Our studies on lens neutral proteinase specificity will be extended to an examination of beta- and gamma-crystallin hydrolysis products and a survey of synthetic proteolytic enzyme substrates.